comparison of four carbon fiber electrodes in microfluidic chip integrated with electrochemical...

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 36, Issue 1, January 2008 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2008, 36(1), 1–6.

Received 26 June 2007; accepted 1 September 2007 * Corresponding author. Email: jkcheng@whu.edu.cn This work was supported by the National Natural Science Foundation of China (Nos. 20405012, 20675060) and the Science fund for Creative Research Group of China (No. 20621502). Copyright © 2008, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.

RESEARCH PAPER

Comparison of Four Carbon Fiber Electrodes in Microfluidic Chip Integrated With Electrochemical Detector Cheng Han, Wu Jian-Hong, Chen Rong-Sheng, Huang Wei-Hua, Wang Zong-Li, Cheng Jie-Ke*

College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, China

Abstract: A microchip electrophoresis system with integrated amperometric detector was developed. Using 0.02 M Tris-HCl (pH = 8) as buffer, at the detection potential of 0.6 V (vs. Ag/AgCl), dopamine (DA) and isoprenaline (IP) were detected by chip-CE with end-column amperometric detection using a carbon fiber microelectrode (CFME), a carbon fiber nanoelectrode (CFNE), a carbon fiber microelectrode modified by single-walled carbon nanotubes (SWNTs-CFME), and a carbon fiber microdisc electrode (CFMDE). The resolution of DA and IP are 0.64, 1.06, 0.61 and 1.22, and the sensitivity of DA (S/N = 3) are 1.7 × 10–7, 5.9 × 10–8, 2.3 × 10–8, and 5.3 × 10–7, respectively. As the CFNE had high resolution and sensitivity, the nanoelectrode-based microchip CE system was successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell was 0.57 ± 0.07 fmol (n = 5), which was in good agreement with that reported in the literatures. Key Words: Microfluidic chip electrophoresis; Electrochemical detection; Dopamine; Isopropyl epinephrine; PC12 cell

1 Introduction

Integrated microchip-based electrophoresis system is a novel electrophoresis technology, which enhances the performance of the chemical analysis with smaller amounts of samples, high analysis speed, high sensitivity, high selectivity, and low cost. Since Manz[1] put forward the new concept of miniaturized total chemical analysis system (μ-TAS) in 1990s, for the past few years, domestic and foreign scholars have carried out many researches related to the exploitation and application of microchip[2–4]. The electrochemical detection method offers the advantages of high sensitivity and selectivity, low sample consumption, low cost, easy integration and miniaturization, which has been widely applied in microchip-based capillary electrophoresis[5–7].

Catecholamines are closely related to activity behavior and environment, and in vivo regulation of human abnormal catecholamine can lead to neurological, psychological, and endocrinological diseases like, e.g., Parkinson’s disease, and Huntington’s disease. The pharmacological actions of catecholamines were deeply studied and used for clinical

purproses. Separation and detection of trace catecholamines in biological sample has significant meaning to study the physiological function, disease diagnosis, drug screening, and curative evaluation. Catecholamines are electrochemically active at proper oxidation potentials and can be detected by microchip capillary electrophoreses with electrochemical detection. The electrochemical detectors especially micro- and nanoelectrodes have many good properties such as higher spatial resolution, the ability to operate in turbid solution, and was widely applied[8,9].

A microchip electrophoresis with end-column electrochemical detection using homemade carbon fiber electrodes (CFME, CFNE, SWNTs-CFME and CFMDE) is presented, the resolution and sensitivity of the four carbon fiber electrodes were compared, and the experiment results showed that the modified electrode had very high sensitivity for determination of DA and IP with a satisfying separation by a carbon fiber microdisc electrode (CFMDE), and the carbon fiber nanoelectrode (CFNE) had higher resolution and sensitivity.

CHENG Han et al. / Chinese Journal of Analytical Chemistry, 2008, 36(1): 1–6

2 Experimental 2.1 Instruments and reagents

Cyclic voltammograms and amperometric determinations were all carried out using a CHI601A electrochemical workstation (CH Instruments, Shanghai, China) in conjunction with a personal computer. A two electrode system was used and a Ag/AgCl reference electrode was used in the experiment. An inverted optical microscope (XDP-1, Shanghai Optical Instrument Factory, China) and a five-dimensional micromanipulation (Shanghai Lianyi Optical Fiber Laser Instrument Factory, China) were used to manipulate the electrode. An XCDY high-voltage supply (0–5 kV, 0–300 mA, four channels, College of Chemistry, Chemical Engineering and Materials Science of Shandong Normal University, China) was used for electrophoresis separation. To minimize the interference of external electronic noise, the whole system was housed in a well-grounded Farady cage.

Dopamine (DA) and isoprenaline (IP) were purchased from Sigma (St. Louis, MO, USA), KLL-8000 acrylic acid cathodic electrophoretic paint was received from Koleal Chemical Co., Ltd. of Wuhan (China), and all other reagents used were of analytical reagent grade or better. Ultrapure water (Water Pro Plus, Labconco, USA) was used to prepare all solutions. Stock solutions (0.01 M) were obtained by dissolving the solid samples in 0.1 M HClO4 and were stored in a refrigerator, and were diluted to the desired concentrations with electrophoresis buffer prior to use. All solutions for the electrophoresis experiments were centrifuged at a rate of 12000 rpm for 10 min to deposit microparticles in the solutions.

2.2 Experimental methods 2.2.1 Fabrication of CFME and CFNE

The methods to fabricate CFME and CFNE were previously described[10]. In brief, a glass (0.9 mm, inner diameter) was pulled on the flame of a gas lamp to form a tip with approximately 20-μm inner diameter. A cleaned carbon fiber (7 μm in diameter, Goodfellow Co., Oxford, U.K.) that was connected to a copper wire with silver print conductive paint (GC Thorsen, 1801Morgan St., Rockford, IL) was inserted from the other end of the glass capillary into the tip. The carbon fiber was then flame-fuse-sealed in the tip of the glass capillary, with about 1-cm length of the carbon fiber exposed from the tip. Within a short time, the capillary tip was fused on the flame to seal the carbon fiber. The CFME was obtained by cutting the protruding carbon fiber to the desired length (about 200 μm) on the inverted microscope, and the CFNE was achieved by etching the protruding carbon on the flame to get a nanometer-scale tip (100–300 nm in diameter, approximately 200 μm in length).

2.2.2 Fabrication of SWNTs-CFME The method to fabricate single-walled carbon nanotubes

(SWNTs-CFME) was previously described by our group[11], and a 3-mg purified SWNTs was dispersed with ultrasonic agitation in 10 ml of aqueous SDS surfactant solution (1 mg ml–1) to obtain a black suspension. The CFME was washed carefully by sonication for 5 min in acetone, alcohol, and double distilled water, respectively. The CFME was then dried under an infrared lamp. The SWNTs suspension was dropped on the tip of the CFME, which was set on a clean planar glass, the protruding carbon fiber was immersed in SWNTs suspension, and the SWNTs-CFME was prepared by evaporating the solution under an infrared lamp for 10 min. The modified electrode was washed carefully by double distilled water before use.

2.2.3 Fabrication of CFMDE

The CFME was cleaned by sonication for 15 s in a sequence

in acetone, alcohol, and double distilled water. A two electrode system was used with Pt electrode as the reference electrode. Stable insulation film was produced by electropolymerization of acrylic acid cathodic electrophoretic paint prepared by amperometric i-t, and the experiment for film deposition was done in the potential of 4 V. The coating thickness was time dependent and 400 s was selected. The CFME was dried under an infrared lamp for 30 min and then placed in an oven at the temperature of 160 °C for one hour, and a compact film was formed on the surface of the CFME. The insulated electrode was washed carefully by double distilled water, and the CFMDE was obtained by cutting the protruding tip of the insulated electrode quickly with a clean scalpel on the inverted microscope.

2.2.4 Microchip CE with end-column electrochemical detector

The schematic illustration of the microchip CE system

mainly composing of microchip for separation and the detection cell is shown in Fig.1. The glass microchip (AMC- µChip-T180, Alberta Microelectronic Corporation, Canada) has a 76-mm-long separation channel and a 250-μm offset double-T injection channel, and the distances from the sample reservoir, buffer reservoir, and sample waste reservoir to the double-T injection channel are all 5 mm. The cross-section of the channels is approximately semicircular with a width of 50 μm at the top and a maximum depth of 20 μm. The original outlet of the buffer waste reservoir was cut to expose the channel outlet and then the edge of the channel outlet was polished with an abrasive. The resulting chip was washed extensively with deionized water and sonicated for 30 min to remove any alumina particles, which may adhere to the

CHENG Han et al. / Chinese Journal of Analytical Chemistry, 2008, 36(1): 1–6

separation or sample channel. Subsequently, the detection cell was formed by adhering a glass slide to the bottom of the chip and another concave-shaped organic glass (poly(methyl methacrylate), 3 mm thickness) on the top of the chip. Finally, an optical microscope and a five-dimensional micromanipula- tor were used to insert the electrode into the separation channel, and then the electrode was fixed on the glass plate by epoxy resin (volume ratio of epoxy resin/ethylenediamine, approximately 8:1), after the epoxy resin was solidified, and a Ag/AgCl reference electrode was positioned sufficiently close to the working electrode and adhered to the detection cell. Pipette tips (200 μl) were cut flat and adhered to the drilled holes (diameter 2 mm) on the chip to form the buffer, sample, and sample waste reservoirs. A platinum wire (diameter 0.5 mm) was inserted into each reservoir as the electrode of high voltage supply. A fast replacement of passivated electrode can be performed by ablating the epoxy with a heated scalpel and refixing a new electrode on the chip.

Fig.1 Schematic diagram of microfluidic chip integrated with

carbon fiber electrode

2.2.5 Treatment of PC12 cell The seeds of PC12 cells were obtained from the American

Type Culture Collection (ATCC) and cultured by the China Center for Type Culture Collection (CCTCC, Wuhan). The growth medium was composed of 85% phenol red-free RPMI-1640 (Gibco, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, NY) and 5% heat-inactivated horse serum (Gibco, NY) in a humidified

atmosphere of 95% air and 5% CO2 at 37 °C. Before the experiment, the PC12 cells were taken out from the culture flask and placed in a centrifuge tube (cubage 10 ml), and centrifuged at the rate of 800 rpm for 6 min to deposit the growth medium. The PC12 cells were washed 5 times with 5 ml physiological salt solution, counted by a cytometer chamber, and lysed in the buffer solution (20 mM Tris-HCl, pH 8.0) containing 0.1% SDS; then the solution was agitated by an ultrasonic generator for 10 min and centrifuged at the rate of 12000 rpm for 10 min to deposit the cell fragments, and the supernatant was used to perform the electrophoresis experiments. The cells were not used over ten passages from the original cell line in the experiments. 3 Results and discussion 3.1 Characterization of electrodes

The cyclic voltammograms of 1 × 10–3 M potassium

ferricyanide in 0.5 M potassium chloride buffer solution obtained with a CFME and a CFNE at a scan rate of 0.1 V s–1

are shown in Fig.2 A and B. Well defined sigmoid-shaped voltammograms are achieved on both CFME and CFNE, which indicate that both the CFME and CFNE display excellent electrochemical characteristics. Figure 2C shows the cyclic voltammograms of a CFME (a), a SWNTs-modified electrode (b), and a CFME modified by the SWNTs twice (c) in potassium ferricyanide solution. The peak current and background current of the modified electrode were much larger than that of the bare CFME; this might be due to the larger geometry and surface area of the SWNTs-CFME. Figure 2D illustrates the cyclic voltammograms by the CFME (a), and the electrode electropolymerised acrylic acid cathodic electrophoretic paint and compact film was formed on it, which was nearly insulated (b). A CFMDE was made by cutting the tip of the insulated electrode with a scalpel on the inverted microscope. Cyclic voltammogram of potassium ferricyanide at a CFMDE was shown as (c), and the result proved that the CFMDE possesses favorable electrochemical behavior.

Fig.2 Cyclic voltammogram curves of carbon fiber electrodes 1.0 mM potassium ferricyanide in 0.5 M potassium chloride buffer solution detected by A. CFME; B. CFNE; C. CFME (a) and SWNTs-CFMES (b, c); D. CFME (a), insulated CFME (b) and CFMDE (c); scan rate, 0.1 V s–1

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3.2 Comparison of detection limit and sensitivity of four carbon fiber electrodes

Figure 3 shows the electropherograms for the separation of 1 × 10–4 M DA and IP in a 20 mM Tris-HCl buffer solution (pH 8) using a CFME (A), a CFNE (B), a SWNTs-CFME (C), and a CFMDE (D), respectively. The resolution of DA and IP and sensitivity for DA were compared with different electrodes. Electrophoresis separation was carried out in uncoated channels that had been flushed with 0.1 M HCl for 10 min, ultrapure water for 10 min, and finally, Tris-HCl buffer for 30 min before microchip CE experiments. All the following experiments were carried out under the optimum conditions: 20 mM Tris-HCl buffer (pH 8.0); injection voltage, 500 V; injection time, 20 s; separation voltage, 1000 V; separation time, 150 s; and detection at 0.6 V. Electrokinetic injection was used for sampling, and the injection voltage was applied to the sample reservoirs with the sample waste reservoir grounded and the other reservoirs floated. Once injection was completed, a separation potential was applied to the buffer reservoir while the detection end was grounded, and then the sample and sample waste reservoirs were floated. In addition, the liquid in the four reservoirs was adjusted on the same horizontal plane to eliminate gravity flow during the experiment.

The microelectrode exhibits well-defined mass transport and rapid response, low IR drop and high S/N, as shown in Fig.3A.

As the size of the electrode reduces to nanoscale, abnormal mass transfer may occur. In addition, the occurrence of quantal phenomena would produce many new excellent electrochemical characteristics, which finally result in the improvement of the high mass-transfer velocity and high resolution[12]. Figure 3B shows that the faster mass transfer and response and less radial diffusion on the nanoelectrodes make the amperometric peaks of the electropherograms sharper and the resolution thus improved compared with that from the microelectrodes. Figure 3C shows amperometric i-t curve of DA and IP obtained by a SWNTs-modified electrode, the electropherogram peak height and background current of the modified electrode is much larger than that of a bare carbon fiber electrode, and the result has a good agreement with the literature[11] and might be due to the modified electrode which has a much larger available internal surface area per external geometric area. So the SWNTs-CFME shows electrocatalytic activity for oxidation of DA and has high sensitivity. At the same time, mass transfer process was limited on SWNTs-CFME, and the resolution was not improved. With CFMDE as working electrode, electronic exchange process performed merely on the disk surface, the peak current was decreased, and sensitivity was decreased too. The samples were detected by the microdisc surface at the exit of the separation channel and no longer were detected as the samples diffuse in the buffer of the detection cell, the peaks width decreased significantly, and DA and IP had better separation.

Fig.3 Electropherograms of DA (1 × 10–4 M) and IP (1 × 10–4 M) using different carbon fiber electrodes Detected by A, CFME; B, CFNE; C, SWNTs-CFME; D, CFMDE. Conditions: running buffer, pH 8.0, 0.02 M Tris-HCl; injection voltage, 500 V; injection time, 20 s; separation voltage, 1000 V; separation time, 150 s; effective separation distance, 76 mm; potential of the working electrode, 0.6 V (vs. Ag/AgCl). Solid arrow, sampling; dotted arrow, separating

The comparison of the resolution of DA and IP, and the

sensitivity for DA using different carbon electrodes (CFME, CFNE, SWNTs-CFME and CFMDE) in the microchip CE-ECD system is shown in Table 1. It can be seen that compared with the CFME, the modified electrode shows higher sensitivity; a better separation between DA and IP was obtained

with CFMDE; and higher sensitivity and better separation efficiency were achieved using the CFNE.

3.3 Analysis of PC12 cells

Figure 4 shows the electropherogram of a PC12 cell extract

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(9.25 × 106 cells ml–1), IP was selected to be the internal standard for the determination of DA in PC12 cell (IP has good peak shape and similar molecular structure with DA), the experiments were repeated for five times, and the content of DA in an individual PC12 cell was 0.57, 0.67, 0.58, 0.54 and 0.49 fmol, respectively. The average content of DA in an individual PC12 cell was calculated to be (0.57 ± 0.07) fmol (n = 5). A comparison of the DA content in PC12 cells determined by CE-ECD reported in the literature with that of this work was drawn. The average content of DA in an individual cell is approximately 0.57 fmol in our work, which is in good agreement with the previous results of our group[13,14]

(approximately 0.54 fmol and 0.61 fmol). However, there are big differences between the results from our work and those of Ewing[15] (approximately 2.9 fmol). Since the same cell lines were used in our laboratory, it was suggested that the differences of DA content in PC12 cell reported by various groups may mainly result from the different origins of cells (different cell lines).

Table 1 Comparison of resolution of DA and IP and sensitivity for DA using different electrodes in the microchip CE-ECD system

Electrodes Resolution Detection limits (M) Electrodes CFME 0.64 1.7 ×10–7 CFNE 1.06 5.9×10–8 SWNTs-CFME 0.61 2.3×10–8 CFMDE 1.22 5.3×10–7

Fig.4 Electropherogram of PC12 cell extract detected by CFNE PC12 cell extract, 9.25 × 106 cells ml–1, using 5×10–6 M IP as the internal standard, other conditions were the same as those in Fig.3 4 Conclusions

A novel and easily operated microchip CE system with

end-column amperometric detection was assembled. The resolution for DA and IP, and sensitivity for DA with four carbon fiber electrodes including CFME, CFNE, SWNTs-CFME and CFMDE were compared, and the experiment result showed that the CFNE had high resolution and sensitivity, and the nanoelectrode-based microchip CE system was successfully used to determine DA in cultured rat pheochromocytoma (PC12) cells. References

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